Postembryonic development of the cranial lateral line canals and neuromasts in zebrafish



The development of the cranial lateral line canals and neuromast organs are described in postembryonic zebrafish (0–80 days postfertilization). Cranial canal development commences several weeks after hatch, is initiated in the vicinity of individual neuromasts, and occurs in four discrete stages that are described histologically. Neuromasts remain in open canal grooves for several weeks during which they dramatically change shape and increase in size by adding hair cells at a rate one-tenth that in the zebrafish inner ear. Scanning electron microscopy demonstrates that neuromasts elongate perpendicular to the canal axis and the axis of hair cell polarization and that they lack a prominent nonsensory cell population surrounding the hair cells—features that make zebrafish neuromasts unusual among fishes. These results demand a reassessment of neuromast and lateral line canal diversity among fishes and highlight the utility of the lateral line system of postembryonic zebrafish for experimental and genetic studies of the development and growth of hair cell epithelia. Developmental Dynamics, 2003. © 2003 Wiley-Liss, Inc.


The mechanosensory lateral line system is a primitive vertebrate sensory system that, like the inner ear, evolved in the ancestor of vertebrates (Northcutt, 1989, 1992). It is found in all fishes and in larval and aquatic adult amphibians, where it allows detection of unidirectional or oscillatory water flow that arise in a variety of behavioral contexts (Montgomery et al., 1995; Coombs and Montgomery, 1999; Modgans and Bleckmann, 2001; Bleckmann et al., 2003), but unlike the ear, it has been lost in amniotes.

The striking similarity in the structure and function of the hair cells of the ear and lateral line system has provided a clear rationale for the exploitation of the lateral line system as a model for the study of the developmental genetics (Whitfield et al., 1996; Nicholson et al., 1998; Itoh and Chitnis, 2001) and regeneration (Balak et al., 1990; Corwin and Warchol, 1991) of the vertebrate auditory system. The morphology of the lateral line system of zebrafish has been described in embryonic and early larval stages (Metcalfe et al., 1985; Metcalfe, 1989; Raible and Kruse, 2000). It has been successfully used to study patterns and mechanisms of migration of placode-derived neuromast precursors and pattern formation of neuromasts on the trunk in zebrafish (Metcalfe, 1985; Smith et al., 1990; Gompel et al., 2001; Sapede et al., 2002; Ledent, 2002; David et al., 2002) and the dynamics of hair cell proliferation and cell death (Williams and Holder, 2000) in embryos and early postembryonic (posthatch) fish (to 5 or 10 days postfertilization [dpf]).

However, it is later during the postembryonic period that the lateral line system of bony fishes begins to take on its adult form with the growth and maturation of neuromasts and the enclosure of presumptive canal neuromasts in pored lateral line canals, which become incorporated in a specific subset of dermatocranial bones (Webb, 1989a, b; Cubbage and Mabee, 1996; Tarby and Webb, 2003). The ontogenetic and morphologic features of the maturing neuromasts and lateral line canals of zebrafish, especially when placed in comparative context, will provide important opportunities to increase our understanding of developmental mechanisms in the lateral line and auditory systems.


The lateral line system of fishes is composed of neuromast receptor organs distributed on the head, trunk, and tail. Two classes of neuromasts are generally present: presumptive canal neuromasts, which are enclosed in canals (canal neuromasts), and smaller superficial neuromasts, which sit in the skin. In juvenile and adult bony fishes, canal neuromasts are found in the epithelium lining the lateral line canals, which are integrated into a subset of dermal bones of the skull (the cranial lateral line canals) and in the lateral line scales on the trunk (trunk canal). Superficial neuromasts are found on the skin surface, may be associated with lateral line canals (e.g., accessory superficial neuromasts, reviewed by Coombs et al., 1988), and may occur in clusters or in linear series. These two types of neuromasts differ in size and shape (Münz, 1989; Webb, 1989c; Tarby and Webb, 2003) and have different biochemical (Song et al., 1995) and functional (Münz, 1989; Kalmijn, 1988, 1989) properties. In some teleosts, nonteleost bony fishes and in amphibians, it has been suggested that heterochronic reduction of canals has resulted in the evolution of a subclass of superficial neuromasts, the “canal replacement neuromasts,” which are homologous to canal neuromasts in related taxa (Coombs et al., 1988). In amphibians, which lack lateral line canals altogether, canal replacement neuromasts, which may proliferate to form linear series called “stitches,” and other superficial neuromasts (“pit organs”) are distinguishable both morphologically and physiologically (Northcutt and Bleckmann, 1993; Northcutt et al., 1994, 1995; Metscher et al., 1997).

Among teleosts, the lateral line canals of the head and trunk vary in morphology and are often used as a source of characters in systematic analyses and phylogenetic reconstructions (reviewed in Webb, 1989b). The morphology of canal neuromasts also varies among species, and appears to be correlated with the morphology of the canals in which they are found (Coombs et al, 1988; Webb, 1989b, 2000b). In narrow lateral line canals, which are well-ossified and uniform in diameter, neuromasts tend to be oval, with the major axis parallel to the canal axis. The hair cells are typically located in an elongate sensory strip that is parallel to the long axis of the neuromast and is generally surrounded by a population of nonsensory support cells, which secrete the gelatinous cupula and determine overall neuromast shape (Webb, 2000a).

In widened canals, which tend to be weakly ossified and nonuniform in diameter, neuromasts are generally much larger than those in narrow canals and have a prominent transverse axis. The sensory strip tends to be round or elongate with the long axis parallel to the canal axis. A nonsensory support cell population surrounds the sensory strip and defines variation in neuromast shape, which may be oval, rectangular (Jakubowski, 1967), diamond-shaped, or in the shape of a robust cross with a prominent transverse axis (Webb, 2000b). These canal neuromasts generally sit in canal constrictions beneath thin bony bridges and the canal roof is typically unossified and takes the form of a tympanum-like epithelium (e.g., Garman, 1899; Jakubowski, 1967; Webb, 1989b, 2000a, b). Widened canals have functional properties that are different from narrow canal systems (Denton and Gray, 1988, 1989; Janssen, 1997). Despite variation in neuromast morphology, the orientation of best physiological sensitivity of the hair cells in canal neuromasts is parallel to the axis of the canal, which allows water moving along the canal axis (induced by pressure differentials at canal pores) to stimulate the hair cells.

In teleost fishes, superficial neuromasts are generally small and round with hair cells distributed throughout the neuromast, but may be diamond-shaped, with hair cells limited to a central region, surrounded by a population of nonsensory support cells, which defines neuromast shape (Webb, 2000a, b). Hair cell orientation in a superficial neuromast is generally related to the body axes (dorsal/ventral; rostral/caudal) or is parallel or perpendicular to the axis of the lateral line canals to which they appear to be accessory (Coombs et al., 1988). With some notable exceptions (e.g., the pit lines of nonteleost bony fishes, Coombs et al., 1988; Webb and Northcutt, 1997), the distribution and morphology of superficial neuromasts are generally overlooked in descriptions of the lateral line system of fishes (Webb, 1989b).


Unlike the inner ear and its innervation, which are derived from a single otic placode, the neuromasts and the sensory neurons that innervate them are derived from a series of dorsolateral placodes on the head (Northcutt, 1989; Northcutt et al., 1994; Baker and Bronner-Fraser, 2001). Migration of placode-derived neurites and neuromast primordia (Metcalfe, 1985; Vischer, 1989), pattern formation, and neuromast differentiation on both the head (Otsuka and Nagai, 1997; Kobayashi et al., 2000) and trunk (Gompel et al., 2001; Sapede et al., 2002; Ledent, 2002) occur during embryogenesis and soon after hatch. Growth and maturation of neuromasts, differentiation of additional (“secondary”) neuromasts on the head and trunk, and the morphogenesis of the lateral line canals occur subsequently (Lekander, 1949; Disler, 1960; Cubbage and Mabee, 1996; reviewed by Webb, 2000b, Tarby and Webb, 2003).

The two classes of neuromasts become distinct during the larval period, as presumptive canal neuromasts increase in size, change in shape, and become distinct from superficial neuromasts, which remain small and round (Münz, 1989; Wonsettler and Webb, 1997; Tarby and Webb, 2003). Morphogenesis of the lateral line canals generally commences during the latter portion of the larval period. Development of the lateral line canals is generally complete by metamorphosis (the transition from the larval to the juvenile stage), which typically occurs several weeks posthatch, but the onset of metamorphosis is quite indistinct in most freshwater fishes, including zebrafish.

Canal formation commences in the vicinity of presumptive canal neuromasts as bony canal walls ossify intramembranously within epithelial ridges forming an open groove in which a presumptive canal neuromast sits. These epithelial ridges then fuse over an individual canal neuromast and the bony canal walls within them fuse forming the bony canal roof of a single canal segment (Wonsettler and Webb, 1997; Tarby and Webb, 2003). Cylindrical canal segments enclosing adjacent neuromasts may grow toward one another leaving one pore between them (Webb, 1989a). The degree of development of lateral line canals varies among species (Webb, 1989a), but as a result of this pattern of development, neuromasts are found in predictable locations between canal pores in the canals of juvenile and adult fishes (Webb and Northcutt, 1997). Despite the diversity in lateral line canal morphology among teleost fishes (see Coombs et al., 1988; Webb, 1989b, 2000a), the lateral line canals are associated with a remarkably consistent subset of dermatocranial bones.

The goal of the present study was to use histologic and scanning electron microscopy (SEM) analyses to describe the pattern of growth and maturation of neuromasts and the pattern of lateral line canal development in postembryonic zebrafish (day of hatch through sexual maturity). Detailed analysis was limited to the supraorbital (SO) canal and mandibular (MD) canal (e.g., the mandibular portion of the preoperculomandibular canal of some authors), which are found on the dorsal and ventral aspects of the skull, respectively. The development of these two canals is particularly conducive to analysis. They are visualized easily with SEM, and whole heads can be embedded in paraffin with accurate orientation so that when sectioned transversely, these linear, cylindrical canals can be accurately visualized. Data on other cranial lateral line canals (e.g., infraorbital, preopercular) and the short trunk canal are presented where available. The results of this work will serve as a baseline for the investigation of mechanisms of development in the lateral line system.


The supraorbital lateral line canal (SO) is uniform in diameter and is contained in the nasal and frontal bones. It contains five neuromasts identified as (SO1–4) in rostral to caudal sequence. A few individuals appeared to have a fifth SO neuromast, but its identity is unclear and is not included in this analysis. Neuromast SO1 is located in the tubular nasal bone, whereas neuromasts SO2–SO4 are found in the portion of the canal in the frontal bone, which forms the roof of the neurocranium (Fig. 1). A terminal pore is present at the anterior end of the portion of the SO canal in the tubular nasal bone. The anterior portion of the frontal bone sits beneath the posterior end of the nasal bone as described by Cubbage and Mabee (1996). The pore at the posterior end of the nasal bone shares an epithelial pore with the anterior terminal pore of the portion of the SO canal in the frontal bone. Caudally, the SO canal is not contiguous with a canal in the bone caudal to it. The canal neuromasts are located between adjacent epithelial canal pores, but the canal roof is not completely ossified in the adult.

Figure 1.

Ontogeny of the canal segments composing the supraorbital (SO) canal in dorsal view. A: Three small depressions (stage IIa) contain SO neuromasts (9 mm standard length [SL], SO1, 2, 3). B: Deeper depressions (stage IIa), especially around SO1 and SO2, 10.5 mm SL. c, cupula. C: Grooves (stage IIb, SO2) and enclosure (stage III, SO1, SO3, SO4), 19 mm SL. Asymmetry in developmental timing is evident in D and E. D: Left SO1, 2, 3 are in grooves (stage IIb), but SO4 is enclosed (stage III). Right SO2 and 3 are in grooves (stage IIb), SO1 and SO4 are enclosed (stage III), 20 mm SL. E: SO1–3 are in grooves (stage IIb); SO4 is in a groove on the left side, but enclosed (stage III) on the right side. A fifth SO neuromast (arrow) is present on the right side, 23 mm SL. F: All canal segments are enclosed (stage III) on both sides of the head, 31 mm SL (adult male; image reversed). Scale bars = 250 μm in A,B, 750 μm in C–F.

The mandibular lateral line canal is uniform in diameter, and is embedded along the length of the dentary and anguloarticular bones. It contains three neuromasts, identified as MD1–MD3 in rostral to caudal sequence (Fig. 2). MD1 and MD2 are contained in the portion of the canal in the dentary bone, whereas MD3 is found in the portion of the canal in the anguloarticular bone. The rostral end of the canal is represented by a terminal pore just to one side of the mandibular symphysis; the left and right canals do not meet at the symphysis. The posterior terminal pore of the portion of the canal in the dentary faces the anterior pore of the short canal portion in the anguloarticular; they share a common epithelial pore. A common epithelial pore is also present at the junction between the mandibular canal and the preopercular canal with which it is contiguous. The canal roof is completely ossified in the adult, and canal neuromasts are located between adjacent canal pores.

Figure 2.

Ontogeny of the mandibular (MD) canal in ventral view. A: Three shallow depressions contain presumptive MD neuromasts MD1–3 in rostral–caudal sequence. Development of canal segments around MD2 and 3 appear to be more advanced (deeper depression) than the segment around MD1, 11 mm SL. ms, superficial neuromasts at mandibular symphysis (ms; see Discussion section in text). B: Asymmetry in timing of development is evident. Right MD1 and 2 are at stage IIb, and MD3 is enclosed (stage III); all three canal segments are enclosed on left, 19 mm SL. C: MD1 is in a groove (stage IIb) and MD2, 3 are enclosed (stage III), 20 mm SL. D: All three canal segments are enclosed (stage III, IV), 31 mm SL (adult male). Scale bars = 150 μm in A, 500 μm in B,D, 250 μm in C.

Pattern of Canal Development

The pattern of morphogenesis of the supraorbital and mandibular canals is defined as a series of four stages (I, IIa/b, III, IV; following Tarby and Webb, 2003; Fig. 3, which describe the development of each canal segment in the vicinity of an individual presumptive canal neuromast. At stage I, a presumptive canal neuromast sits on the flat epithelium above a thin underlying dermal bone (Figs. 4A, 5B). At stage IIa (Figs. 4B, 5C), the neuromast sits in a shallow epithelial depression. At stage IIb (Figs. 4C, 5D), the presumptive canal neuromast appears to have sunken into a deep canal groove as a pair of parallel walls ossify intramembranously, rising from the bony floor of the canal on either side of the neuromast. Stage III (Figs. 4D, 5E) is defined by the enclosure of the neuromast by the fusion of the soft tissue walls of the groove forming an epithelial canal, and stage IV is defined by the fusion of the two bony canal walls within the epithelial canal to form an ossified canal roof (Fig. 5F).

Figure 3.

Schematic representation of developmental stages of the canal segments in the mandibular and supraorbital canals of the zebrafish Danio rerio. Stage I: Presumptive canal neuromast on the epithelium and above thin underlying dermal bone. Stage IIa: Neuromast sits in a shallow depression. Stage IIb: Neuromast sits at base of deep open canal groove. Ossified bony walls extend upward from underlying bone. Stage III: Canal is enclosed by epithelium that fuses over neuromast. Stage IV: Ossified canal walls fuse over the neuromast to form a canal segment with ossified canal roof.

Figure 4.

Development of the supraorbital (SO) canal at neuromast SO1. A: Presumptive canal neuromast (nm) sitting on the epithelial surface (stage I), underlying dermal bone (pink), and embryonic cartilage (blue), 10 mm standard length (SL). B: Neuromast (nm) in epithelial depression (stage IIb) with underlying dermal bone (pink), 10 mm SL. C: Neuromast in canal groove (stage IIb) bounded by ossified canal walls (cw; 12 mm SL) D: Neuromast with hair cells wrapped around circumference of canal (arrows) enclosed by epithelial roof (22 mm SL). Neuromasts in A, C, and D clearly show layer of hair cells and layer of support cells beneath hair cells. Scale bars = 50 μm in A–D.

Figure 5.

Development of the mandibular (MD) canal. A: Neuromasts (nm) in epithelium (stage I), overlying dermal bone (pink) surrounding Meckel's cartilage (blue), 11. 5 mm standard length (SL). B: Neuromast (nm) at bottom of epithelial depression (stage IIa), 11 mm SL. C: Neuromast in canal groove (stage IIb) with ossified canal walls (pink, cw) extending toward the surface, 20 mm SL. D: Neuromast in canal enclosed by epithelial roof (er) with ossified canal walls (pink; stage III), 12 mm SL. E: Neuromast enclosed in canal with ossified canal roof (pink, cr), 22 mm SL. Scale bars = 50 μm in A–E.

Timing of Canal Development

Development of the different canal segments that compose a canal, and of the different canals, is asynchronous. Development of the SO canal commences in individuals 10–11 mm standard length (SL; ∼30–32 dpf; Tables 1, 2). However, short grooves are evident in SEM scans of individuals as small as 9 mm SL (∼24 dpf), which are otherwise obscured by copious mucus (Fig. 1A). Stage IIa is not documented for all canal segments, which is indicative of its short duration (Table 1). At 12 mm SL (∼36 dpf), all canal neuromasts sit in canal grooves with ossified canal walls (stage IIb, the “bony struts” of Cubbage and Mabee, 1996). This stage generally persists in fish of up to 20–23 mm SL (∼70–80 dpf). Both SEM and histologic material demonstrate that all SO canal segments are at stage III in adult fish (Fig. 1F, Table 1)—canal roof ossification is not complete, therefore, stage IV is never observed.

Table 1. Development of Supraorbital and Mandibular Canal Segmentsa
  • a

    Derived from histological series (TAB-5 wild-type, 8.5–22.0 mm SL, n = 12; EK wild type, n = 1, 31 mm SL). Each row represents data taken from one individual. SO, supraorbital; MD, mandibular; SL, standard length; L, left; R, right.

Table 2. Timing of Lateral Line Canal Development Based on SEM and Histological Analysisa
CanalDepressions presentGroove formation beginsCanal enclosure beginsCanal enclosure complete
  • a

    All fish sizes are in mm standard length (SL). Ages (dpf) are approximate (see Fig. 11). See also Table 1 for data on SO and MD canals. SEM, scanning electron microscopy; dpf, days postfertilization; SO, supraorbital; MD, mandibular.

  • bData from Cubbage and Mabee (1996).

  • c

    Data from SEM only.

 IO1 (lacrimal)??7 mm (17 dpf)*16 mm (38 dpf)
 IO2-5?15.5 mm (38 dpf)16 mm (38 dpf)>23 mm (>72 dpf)
Preopercularc?<11 mm (<28 dpf)11 mm (28 dpf)15.5 mm (38 dpf)
Supraorbital10 mm (30 dpf)11 mm (28–36 dpf)16–21 mm (38–64 dpf)>23 mm (>72 dpf)
Mandibular11 mm (28 dpf)11–12 mm (28–36 dpf)16–21 mm (38–64 dpf)21–>23 mm (64–>72 dpf)

Development of the MD canal generally commences in 11 mm SL (∼32 dpf) fish when presumptive canal neuromasts can be seen sitting in longitudinal depressions (stage IIa; Tables 1, 2; Fig. 2). Stage IIa is evident in most histologic series for no more than a 0.5-mm growth interval and may be absent in some canal segments in some individuals, suggesting its short duration (Table 1). Stage IIb is the stage of longest duration, lasting for several weeks, and is seen in animals up to 18–20 mm SL (∼60–70 dpf). Canal enclosure (stage III) generally occurs in individuals 20–21 mm SL (∼70–72 dpf). Canal roof ossification (stage IV) occurs quickly thereafter, such that stage III is not recorded for some canal segments (e.g., MD1, 3). Stage IV is the terminal condition for all canal segments of the mandibular canal.

The development of segments in the SO and MD lateral line canal series occur asynchronously and somewhat asymmetrically (between homologous canal segments on the right and left sides; Table 1; Figs. 1, 2). Histologic analysis could not define any clear directional trends in the development of segments within a canal, but SEM data suggest a caudal to rostral trend in the MD canals, in which the MD1 neuromasts are the last to enclose (Fig. 2C).

The other cranial lateral line canals develop before or after the SO and MD canals (Table 2). SEM data clearly demonstrate that the preopercular canal encloses early and quickly (by 15.5 mm SL; 38 dpf). The portion of the infraorbital canal in the lacrimal bone is also enclosed early (16 mm SL, ∼38 dpf), and the remainder of the infraorbital canal (located in infraorbital bones 2–5) is the last of the canals to enclose (> 23 mm SL; > 72 dpf; Fig. 8). Thus, the complete development of all of the cranial lateral line canals, from the commencement of groove formation (stage IIa) through canal enclosure (stage III) and the ossification of the canal roof (stage IV, except the SO canal, whose development is terminated at stage III), occurs over a period of more than 1 month.

Developmental Morphology of Canal Neuromasts

At hatch, neuromasts are visible in the vicinity of the future SO canal, but presumptive canal neuromasts cannot be distinguished from superficial neuromasts in either histologic material or in SEM scans. At this point, all of the neuromasts are small and round, measuring approximately 5–10 μm in diameter. A pair of superficial neuromasts is clearly present at the mandibular symphysis in 3-mm individuals (interpreted to be neuromast M1 of Raible and Kruse, 2000) and a cluster of superficial neuromasts is seen in this location in older individuals (Fig. 2A). Presumptive canal neuromasts SO1–4 are evident in SEM scans of a 5-mm (∼8 dpf) individual and sit on the flat epithelium without evidence of any canal depressions. In individuals between 5 and 11 mm (∼8–32 dpf), there is a dramatic increase in the density of mucus-producing cells that limits the ability to visualize neuromasts without sonication. In 5- to 6.5-mm individuals (∼9–14 dpf), all of the presumptive canal neuromasts of the supraorbital canal (SO1–4), but only one in the mandibular canal series (MD2) is present. MD1 and 3 appear later in individuals 6.5–11 mm SL (∼14–32 dpf). In 6.5-mm individuals (∼14 dpf), both superficial and presumptive canal neuromasts overlie the infraorbital bones, dentary, anguloarticular, and preopercular bones, and additional lines of superficial neuromasts overlie other bones (e.g., the opercular).

Presumptive canal neuromasts of both the SO and MD canal series could be clearly distinguished from superficial neuromasts in SEM scans of individuals as small as 9.0 mm SL (∼24 dpf, Fig. 6) based on their topography and the morphology of the surrounding epithelial cells. Presumptive canal neuromasts sit flat in the epithelium, whereas superficial neuromasts are slightly papillate, with their sensory epithelium sunken beneath the edge of the neuromast. Interestingly, weak microvilli are observed on the surface of the epithelial cells surrounding presumptive canal neuromasts (Fig. 6A), whereas strong microvillar ridges are present on the surface of the epithelial cells surrounding superficial neuromasts (Fig. 6B).

Figure 6.

Two classes of neuromasts in a 9 mm standard length (SL) individual. A: Presumptive supraorbital (SO) canal neuromast (SO2) with hair cell epithelium demonstrating very long kinocilia, sitting flat in epithelium, with weak microvillar ridges, which are largely obscured by large flakes of dehydrated mucus. B: Superficial neuromast with very long kinocilia, sitting on a small papilla, surrounded by epithelial cells with strong microvillar ridges. Double-headed arrows show hair cell orientation. Scale bars = 5 μm in A,B.

Despite variation in the timing of canal development among canal segments, canals, and individuals, the developmental morphology of individual canal neuromasts appears to be correlated with the stage of development of the canal segment with which it is associated. Presumptive SO canal neuromasts are found in short canal depressions, indicating the commencement of canal development. As a canal depression (stage IIa) develops into a canal groove with ossification of canal walls (stage IIb), the presumptive canal neuromast starts increasing in size and changing shape (Fig. 7), becoming quite distinct from superficial neuromasts, which remain small and round (Figs. 8, 9). Over several weeks, the length (major axis) to width (minor axis) ratio of presumptive canal neuromasts increases dramatically (e.g., from 1:1 to ∼10:1 or more, Fig. 9), with little increase in width. This results in a change in neuromast shape from round to narrow and elongate, with the long axis of the neuromast transverse to the canal axis (Fig. 10). This explains why canal neuromasts appear to “wrap” around the floor and walls of the canal in transverse sections in adult zebrafish (e.g., Fig. 4D). Despite the transverse placement of the elongate neuromast, their hair cells are polarized with the axis of best physiological sensitivity parallel to the canal axis, thus allowing water movement along the canal axis to provide an effective hair cell stimulus. As canal grooves develop, there appears to be a reduction in the microvillar ridges in the epithelial lining of the groove (see Fig. 7). This finding may be an adaptive feature because the presence of strong microvillar ridges would retain surface mucus (Sperry and Wassersug, 1976), which would likely increase viscosity, impede water movement, and reduce the sensitivity of the neuromasts sitting in grooves or enclosed in canals.

Figure 7.

Ontogenetic shape change of a mandibular (MD) canal neuromast (MD3). A: Neuromast located in a shallow depression (stage 11a), 11 mm standard length (SL). B: Close-up of neuromast in A, showing round shape. C: Neuromast in groove (stage 11b), 12 mm SL. D: Close-up of neuromast in C showing ovoid shape with long axis transverse to canal axis. Double-headed arrows in B and D indicate axis of best physiological sensitivity (orientation) of hair cells, which appears to remain consistent as the neuromasts change morphology. Scale bars = 25 μm in A,C, 5 μm in B,D.

Figure 8.

Two classes of neuromasts on the head of a 20 mm SL zebrafish. A: Lateral view of head. The infraorbital canal (just caudal to orbit) is an open groove, whereas preopercular, mandibular canals and the portion of the infraorbital canal in the lacrimal bones are enclosed with prominent canal pores. B: Presumptive canal neuromast in infraorbital canal at arrow 1 in A. C: Superficial neuromast on posterior edge of operculum at arrow 2 in A. Scale bar = 500 μm in A, 10 μm in B, 5μm in C.

Figure 9.

Ontogeny of size and shape of presumptive supraorbital (SO) canal neuromasts in the supraorbital (SO1–3, filled circles, solid line) and mandibular (MD1–3, open circles, dashed line) canals and superficial neuromasts (crosses, dotted line) in postembryonic zebrafish (3–23 mm standard length [SL]). Regressions for SO neuromasts are provided for context. A: Length of neuromasts vs. fish size. Length = dimension transverse to canal axis (long axis of the neuromast). Width = dimension parallel to canal axis (short axis of neuromast). SO neuromasts: y = 12.053x − 104.12, r = 0.83. B: Length to width ratio vs. fish size. Arrow indicates the fish size at which neuromasts first exceed a 1:1 ratio (= 11 mm SL). Symbols represent neuromasts with 1:1, 3:1, and 10:1 ratios (grey area indicates distribution of hair cells). Variation in the size and shape of neuromasts associated with different canals is further explored in Table 1. SO neuromasts: y = 0.767x − 6.7, r = 0.81. C: Hair cell number in individual neuromasts vs. fish size. Some hair cells were obscured by long kinocilia and could not be counted; therefore, these values should be considered minimum hair cell counts. SO neuromasts: y = 5.079x − 28.46, r = 0.87.

Figure 10.

Ontogeny of presumptive canal neuromasts in the supraorbital (SO) canal. A: Neuromast SO3 in a 9 mm individual. Kc, kinocilium. B: SO3 in a 15 mm individual. C: SO3 in 23 mm individual. D: Dorsal view (rostral to left) of SO canal groove (23 mm standard length) indicating the location of neuromasts SO1–4. E: Close-up of canal groove indicating the location of SO1. Scale bars = 5 μm in A, 10 μm in B, 35 μm in C, 250 μm in E.

The elongate, transverse canal neuromast morphology described in the SO and MD canal series is also characteristic of the presumptive canal neuromasts of all canal series on the head (Table 3; unpublished observations), as well as the presumptive canal neuromasts that become enclosed in the few tubular lateral line scales that form the short lateral line canal on the trunk (Table 3). The SO and MD canal neuromasts demonstrate the most exaggerated elongation, traversing the entire width of the canal groove, and attaining a length of up to 200 μm or more before becoming enclosed (Fig. 10; Table 3). The infraorbital canal neuromasts are much smaller than the SO neuromasts in a given individual; they have a smaller length to width ratio and a slower rate of increase in length (data not shown), which is correlated with a delay in the development of the infraorbital canal relative to other canals (Fig. 8). The elongate, transverse neuromast morphology reported here for the TAB-5 wild-type fish is present in another wild-type strain (EK). In addition, this morphology is found in the F1 of field-caught animals; the neuromasts are transversely placed, albeit a bit shorter and wider, but have more hair cells (>100) than homologous neuromasts in a wild-type individual of the same size (20 mm SL, data not shown), indicating that this overall neuromast morphology is a characteristic of the species.

Table 3. Variation in Canal Neuromast Size, Shape, and Hair Cell Number for SO (SO1–3), IO, and Trunk Canal Neuromasts and Superficial Neuromasts in Two Individuals of the Same Size and Age (76 dpf, 20 mm SL)a
 Supraorbital canal neuromasts (n = 3)Infraorbital canal neuromasts (n = 4)Trunk canal neuromasts (n = 2)Superficial neuromasts (n = 4)
  • a

    Mean values (in μm) and standard deviations are calculated from measurements taken directly from SEM scans. Neuromast lengths are likely underestimated due to the curvature of the canal lumen. SO, supraorbital; IO, infraorbital; dpf, days postfertilization; SL, standard length; SEM, scanning electron microscopy.

Length173.0 ± 23.645.8 ± 4.127.512.0 ± 4.1
Width13.0 ± 2.68.25 ± 0.968.510.8 ± 3.0
L/W ratio13.7 ± 4.75.75 ± 0.963.51.0 ± 0

As zebrafish grow, canal neuromasts increase in size and elongate perpendicular to the canal axis with a concurrent increase in hair cell number (Fig. 9C). For instance, 13–20 hair cells are present in each of the SO canal neuromasts in a 9 mm SL individual, and 72–78 hair cells are found in these neuromasts in a 20 mm SL individual. In addition, canal neuromasts in the MD series appear to increase in size, change shape, and add hair cells at a rate that is less than half that of the SO neuromasts in the same individual. The hair cells are oriented 180 degrees to one another and are distributed throughout the long, narrow neuromast epithelium, which has a distinct outer boundary that abuts the general epithelium in which the neuromast sits (see Figs. 8B, 10B,C). Only one or two nonsensory cells surround the hair cell population; a prominent population of nonsensory support cells, which surrounds the hair cell population in neuromasts of other species, is not present.

Superficial neuromasts increase in number with fish size, but they remain small (<16 μm) and round (∼1:1 length:width ratio), with a small number of hair cells (<20, Figs. 8, 9) oriented 180 degrees to each other. SEM scans reveal that the length of the kinocilium of the hair cells in canal and superficial neuromasts is generally between 6 and 12 μm, but that the kinocilia of superficial neuromasts appear to be longer than those in canal neuromasts in the same individual.


The present study extends our understanding of the development of the lateral line system of zebrafish through the postembryonic period to sexual maturity. Histologic and SEM analyses provide an opportunity for the simultaneous description of neuromast morphology, distribution and ontogeny, and the pattern of lateral line canal morphogenesis. Our results show that the general morphology and pattern of development of the cranial lateral line canals in zebrafish is similar to that reported in other teleosts (Webb, 1989a; Tarby and Webb, 2003), but that the timing of canal development relative to the growth and maturation of canal neuromasts, neuromast morphology, and the relationship of neuromast to canal morphology, in zebrafish appear to be unusual among fishes.

Pattern and Timing of Canal Development

The pattern of development of the supraorbital and mandibular canals in zebrafish is almost identical to that recently reported for the cichlid, Archocentrus nigrofasciatus (Tarby and Webb, 2003). Canal development proceeds through four stages, but the formation of soft tissue depressions (stage IIa) and grooves with ossified canal walls (stage IIb) are clearly visible in the zebrafish, whereas stage IIa could not be detected in A. nigrofasciatus (Tarby and Webb, 2003).

The timing of canal development in zebrafish clearly differs from that in Archocentrus nigrofasciatus. Canal morphogenesis in A. nigrofasciatus, is initiated in small posthatch individuals (6.0–6.5 mm SL), and neuromasts sit in grooves for just a short period of time (growth interval of 0.5–1.5 mm); canal enclosure (stage III) occurs quickly (at 6.5–11.0 mm SL) as canal neuromast length increases (in stages II–IV; Tarby and Webb, 2003). In zebrafish, neuromasts sit in the skin (stage I) for the first month of postembryonic development without any sign of canal morphogenesis. Shallow canal depressions (stage IIa) become apparent on the dorsal surface of the head (SO canal) and on the mandible (MD canal) in 10–11 mm SL individuals (∼28–30 dpf). The presumptive canal neuromasts of the supraorbital and mandibular series remain in open grooves (stage IIb) for more than a month. The delay of canal enclosure relative to initiation of neuromast elongation in zebrafish affords an opportunity to directly observe changes in the size and shape of the sensory epithelium in canal neuromasts. Furthermore, variation in the timing of the onset and duration of the different stages of canal development can provide a context in which to analyze the evolution of developmental patterns in the lateral line system among fishes.

Despite asynchrony in the development of different canal segments within and among canals, and variation in the duration of different stages of canal development, the maturation of individual canal neuromasts (e.g., initiation of elongation) in zebrafish appears to be correlated with the development of the canal segment in which it sits. At stage IIa, canal neuromasts are round, but as grooves form and canal walls ossify (stage IIb), neuromasts start to elongate transversely with a rapid increase in hair cell number. This finding provides evidence for a developmental interaction between canal neuromasts and dermal bone composing the lateral line canals, which has yet to be characterized (discussed by Hall and Hanken, 1985; Wonsettler and Webb, 1997).

Developmental Integration of Lateral Line Canals and Dermatocranial Bones

The way in which the lateral line canals become integrated into the “lateral line” bones is reported to vary among fishes (reviewed in Tarby and Webb, 2003). In some fishes, the dermatocranial bones containing the lateral line canals develop as the result of the fusion of two centers of ossification—the lateral line canal and an underlying dermal bone. A consideration of the phylogenetic distribution of those fishes in which such a “two-component” pattern of lateral line bone development suggests that there might be a phylogenetic trend from a two-component pattern in more basal teleosts (e.g., ostariophysans, including zebrafish) to a “one-component” patterns in more advanced teleosts (e.g., cichlids), where the walls of the lateral line canals extend upward from the underlying bone (discussed in Tarby and Webb, 2003). However, the results of the present study demonstrate that the SO and MD canals of zebrafish exhibit a one-component pattern of development, which is remarkably similar to that in the cichlid, Archocentrus nigrofasciatus (Tarby and Webb, 2003). There is no evidence of a two-component pattern of development in the supraorbital or mandibular canals in zebrafish. Two explanations are offered to account for this observation.

First, zebrafish may be atypical of other ostariophysans, whose lateral line bones have been reported to have a two-component pattern of development (Lekander, 1949; discussed by Tarby and Webb, 2003). The unusual neuromast morphology and the unusual association of transversely placed neuromasts in a narrow canal system in zebrafish, appear to be rare among fishes. If an alteration in the pattern of development of the lateral line canals relative to the underlying dermal bones accompanies the evolution of this specialized morphology, this may provide an explanation for the presence of a one-component pattern of lateral line bone development in zebrafish. This pattern would be particularly interesting, because the zebrafish, Danio rerio, one of several thousand members of the Family Cyprinidae (true minnows), does not appear to have any obvious specialized characteristics and could otherwise be described as a cyprinid with a “generalized” morphology.

An alternative explanation is that, while the supraorbital and mandibular canals in zebrafish demonstrate a one-component pattern of development, the other components of the cranial lateral line canal system (contained in the parietal, posttemporal, pterotic, and supratemporal bones; Cubbage and Mabee, 1996) may demonstrate a two-component pattern of development. The variation in the association of lateral line canals with particular dermal skeletal elements observed among individual zebrafish (Cubbage and Mabee, 1996) and among species (e.g., nonteleost bony fishes), suggests some degree of independence of the development of lateral line canals from the underlying dermal bones, so we speculate that this second explanation is more likely. However, a more extensive, comparative analysis of lateral line canal development among canals in zebrafish, and of homologous skeletal elements among carefully chosen species, will be necessary to evaluate this hypothesis.

Establishing Neuromast Identities in Zebrafish

In adult fishes, the identity of individual canal neuromasts can be defined by their innervation by branches of the lateral line nerves (Coombs et al., 1988; Northcutt, 1989, 1992) as well as by their position in a lateral line canal and the identity of the dermal bone with which the canal is associated (Tarby and Webb, 2003; this study). In contrast, neuromasts in fish embryos and larvae have been mapped by using fluorescent markers and have been named according to both their location and innervation (Raible and Kruse, 2000) or by inferring their location based on the placement of foramina that carry small branches of the lateral line nerve (Cubbage and Mabee, 1996). However, in embryonic and early larval stages, presumptive canal neuromasts and superficial neuromasts cannot be distinguished so this poses interesting problems for the establishment of neuromast identities.

For instance, by using SEM and histology, we identified four neuromasts in the supraorbital canal series (501–504) in zebrafish larger than 12 mm SL. Cubbage and Mabee (1996) illustrate only two pairs of “bony struts” (ossified canal walls) in the frontal bone of a 15.2 mm individual cleared and stained for ossified bone. Based on their location, we interpret these to be the canal walls associated with neuromasts SO2 and SO4; the ossified canal walls associated with SO3 are not documented. In addition, the number of nerve foramina associated with both the SO and MD canals (Cubbage and Mabee, 1996), exceeds by one the number of canal neuromasts we have identified in these canal series using SEM. We suggest that, in each case, the “extra” foramen carries a nerve branch to nearby superficial neuromasts or carries small blood vessels that supply the capillary beds that underlie neuromasts (illustrated by Jakubowski, 1963, 1967). Thus, the number of foramina in lateral line bones cannot be used to predict the number of canal neuromasts in larval zebrafish, or perhaps other larval fishes.

The mandibular neuromast M1 identified by Raible and Kruse (2000) in 4 dpf zebrafish appears not to be one of the three presumptive canal neuromasts in the MD series (our MD1–3). Instead, we interpret this to be the first of several superficial neuromasts clustered at the mandibular symphysis (see Fig. 2A). We have also demonstrated that two of the three presumptive canal neuromasts of the MD series (MD1 and 3) appear later than either MD2 or the superficial neuromasts at the mandibular symphysis. This finding is particularly interesting because it demonstrates that, in zebrafish (and perhaps in other teleosts), primary neuromasts (those that differentiate early) are not necessarily presumptive canal neuromasts and that secondary neuromasts (differentiate later) are not necessarily superficial neuromasts. Furthermore, Raible and Kruse (2000) identify three SO neuromasts in 4 dpf fish, which we interpret as presumptive canal neuromasts SO1–3. However, they do not document the presence of our SO4. Instead, they identify neuromast O1 just caudal to SO3 and indicate that it is innervated by a different nerve branch than the SO neuromasts. However, our SEM scans indicate that our SO4, which is clearly integrated into the SO canal (but is of unknown innervation) is smaller than SO1–3 and that the development of the canal segment around SO4 lags behind that of SO1–3 (Table 1). Thus, we suggest that SO4 is part of the SO series and that it differentiates later than 4 dpf and that it is not neuromast O1.

Given such discrepancies with other recent studies, we suggest that a combination of methods (e.g., SEM, histology, fluorescent markers, cleared and stained osteologic preparations) that can simultaneously track neuromast and canal morphology and nerve innervation needs to be used to establish neuromast identities at several time points through postembryonic development, as innervation patterns become more complex with the asynchronous differentiation of presumptive canal and superficial neuromasts and the enclosure of canal neuromasts in the lateral line canals.

Unusual Neuromast and Canal Morphology in Context

The elongation of neuromasts perpendicular to the axis of best physiological sensitivity of the hair cells, the absence of a population of nonsensory cells surrounding the sensory strip, and the occurrence of transverse neuromasts in what appear to be narrow lateral line canals (Webb, 1989b) are interesting and unusual morphologic attributes of zebrafish canal neuromasts, which only become apparent several weeks posthatch and have escaped notice until now.

The elongate, transverse neuromast morphology in zebrafish appears to be rare among cyprinids (for which little is known about neuromast morphology) and among teleost fishes. Transverse neuromasts are not found in the other important cyprinid model species, the goldfish (Carassius auratus, Puzdrowski, 1989; personal observation), which has neuromasts typical of narrow canal systems that are round or oval with the long axis parallel to the canal axis. However, the North American cyprinid, Notropis buccatus (= Ericymba buccata, the silverjaw minnow) has narrow, elongate, transversely placed neuromasts, which lack a prominent nonsensory population surrounding the hair cells, and are thus identical to the canal neuromasts of zebrafish (Webb and Herman, unpublished observations; contrary to illustration in Reno, 1971). The neuromasts of another group of teleost fishes, the percids (Order Perciformes), demonstrate a great deal of variation in neuromast shape, placement, and the relative sizes of the sensory and nonsensory cell populations (Jakubowski, 1967). Several percid species are described as having transverse neuromasts, and two of these have elongate oval or rectangular neuromasts with very narrow nonsensory cell populations surrounding the hair cell population (Acerina acerina and Aspro zingel, Jakubowski, 1967). A more extensive survey of neuromast morphology among fishes is clearly needed in order to put zebrafish in context.

Despite the atypical nature of the morphology of zebrafish canal neuromasts, they can be exploited as a system in which to address some fundamental developmental issues. The axis of hair cell polarization is generally parallel to the long axis of the sensory strip of canal neuromasts, which is in turn parallel to the canal axis (Rouse and Pickles, 1991a). However, our data from postembryonic zebrafish indicate that the long axis of the canal neuromasts is placed perpendicular to the axis of hair cell polarization. Thus, we suggest that the canal neuromasts of zebrafish will provide an interesting context in which to analyze the dynamics of hair cell differentiation and proliferation, especially with respect to the relationship of the axes of mitosis, cell division, and hair cell polarization and resultant changes in the shape of hair cell epithelia.

The rate of addition of hair cells in the zebrafish ear is ∼8 hair cells/day in animals ∼3.5–7 dpf (anterior and posterior maculae, calculated from data in Haddon and Lewis, 1996). The rate of addition is ∼8 hair cells/day in the utricle and sacculus, and ∼12 hair cells/day in the lagena of animals 5–30 dpf (calculated from data in Bang et al., 2001). In juvenile and adult zebrafish (up to 7 months old), the rate of hair cell addition is approximately the same (∼10 hair cells/day in the sacculus and ∼13 hair cells/day in the lagena; Higgs et al., 2003). In stark contrast, the rate of addition of hair cells in the SO canal neuromasts of zebrafish up to 3 months old is only 0.93 hair cells/day (see Fig. 9). Thus, the rate at which hair cells are added to the sensory maculae of the inner ear is 10 times the rate at which hair cells are added to the supraorbital neuromasts in zebrafish. Rates of hair cell turnover, which are only known for neuromasts in 10 dpf zebrafish (Williams and Holder, 2000), which still retain their round shape; rates of hair cell differentiation and turnover have yet to be investigated in maturing (elongating) neuromasts in postembryonic zebrafish. This slower rate of hair cell addition in neuromasts provides an opportunity to compare the dynamics of hair cell populations in two vertebrate sensory systems.

The canal neuromasts of fishes other than zebrafish generally have a prominent population of nonsensory support cells that surround the sensory strip, which is generally round or elongate, in the axis of the canal (Webb, 2000a). The size and shape of this nonsensory cell population define the considerable variation in neuromast shape found among fishes. In embryonic and early larval zebrafish, hair cells are distributed throughout the small, round neuromast (Williams and Holder, 2000; this study). As presumptive canal neuromasts elongate, hair cells increase in number, presumably differentiating from the support cells that are scattered among the hair cells and also located in a narrow ring (one to two cells) surrounding the hair cell population. Hair cells are added at the periphery of neuromasts in 10 dpf zebrafish (Williams and Holder, 2000) and, thus, likely arise from this narrow ring of support cells in older embryos and larvae. However, the proliferation of nonsensory support cells that is necessary to generate the prominent nonsensory cell population characteristic of canal neuromasts in other fishes appears not to occur in zebrafish. The absence of this large nonsensory cell population means that zebrafish canal neuromasts are more similar to the sensory epithelia of the inner ear of zebrafish than they are to the neuromasts of other fish species. This observation raises interesting questions about the temporal and spatial expression patterns of genes that determine the proportion of hair cells and support cells in sensory epithelia of both the lateral line and inner ear.

The correlation between neuromast and canal morphology has been noted in several studies. Narrow lateral line canal systems, which are described as being uniform in diameter, generally contain round or oval canal neuromasts whose major axis is parallel to the canal axis. In contrast, widened canal systems are generally non-uniform in diameter and contain neuromasts (of various shapes among species), which have a prominent transverse axis and sit in periodic constrictions along the canal (reviewed by Webb, 2000a, b). Widened canals also tend to have a membranous canal roof, which is generally ossified only in the vicinity of each canal neuromast (e.g., several percids, Jakubowski, 1967; reviewed by Webb, 1989b; Webb 2000a, b). We have demonstrated that the cranial lateral line canals in zebrafish are generally uniform in diameter, but contain elongate, transversely placed neuromasts. Besides this unusual neuromast morphology, the association of neuromasts with a prominent transverse axis with a narrow (and in most cases, well-ossified) canal system, challenges the notion that neuromast morphology is correlated with canal morphology among species (see Coombs, et al., 1988; Webb, 1989b, 2000a, b). It is not known whether this association of transverse neuromasts with a narrow canal system is a feature of cyprinid fishes other than zebrafish, because while the canal systems of cyprinids have been described in some detail (e.g., Illick, 1956; Reno, 1966; Gosline, 1974; Mayden, 1989), the morphology of the neuromasts in this diverse taxon has not been studied. However, one cyprinid (Notropis buccatus [=Ericymba buccata], Reno, 1971; Hoyt, 1972) has widened canals, of uniform diameter, which contain transverse neuromasts that are virtually identical to those of zebrafish. Thus, transverse neuromasts are found in both narrow, well ossified canals and in widened, weakly ossified canals of uniform diameter. In addition, a growing number of phylogenetically diverse taxa (e.g., Notopterus, Omarkhan, 1948; Eigenmannia, Vischer, 1989; Cataetyx, Gibbs, 1999) have been reported to have neuromasts with a prominent transverse axis, located in widened canals with a weakly ossified canal roof and a uniform diameter. The breakdown of the reported correlation between neuromast and canal morphology among fishes suggest that lateral line canal and canal neuromast morphology have evolved more independently of one another than previously thought. Analyses of the pattern and relative timing of neuromast and canal ontogeny among species with divergent adult canal morphologies, coupled with analyses of tissue interactions during neuromast maturation and canal morphogenesis, will be necessary to identify the developmental mechanisms underlying the evolutionary diversification of the lateral line system.

The zebrafish continues to provide an extraordinary amount of insight into fundamental aspects of vertebrate embryogenesis. The unusual canal neuromast morphology that we have described in postembryonic zebrafish can provide a new context for the analysis of the genetics of hair cell differentiation and hair cell polarization, and the dynamics of growth in hair cell epithelia. The accessibility of the hair cells of the lateral line system, which are found in small, discrete populations on the external surface of the head for several weeks, can provide interesting opportunities for the imaging of developing sensory epithelia and will provide a useful comparison with developmental patterns and processes in the inner ear. The development of lateral line canals and their relationship to underlying dermal bones will likely provide an interesting context for the analysis of tissue interactions in the development of the dermatocranium, especially when placed in a comparative context. So, despite the irony that the postembryonic lateral line system of this model species appears not be typical of teleost fishes (see Bolker, 1995; Bolker and Raff, 1997; Metscher and Alhberg, 1999), this species will continue to provide important insights because it has broadened the morphologic context in which patterns and mechanisms of development and evolution in the lateral line system and inner ear may be interpreted.


Study Animals

TAB-5 wild-type fish were reared at 26°C (maximum range of 25–29°C) with a 13 hr/11 hr light–dark cycle using standard methods. Fish were fixed in 10% formalin in phosphate buffered saline (PBS) and stored at 4°C. Four individuals were collected daily from 2 to 22 dpf, and every other day from 22 to 80 dpf to generate a growth series from hatch through sexual maturity. Several months after fixation, the notochordal or standard length (SL) of each fish was measured before further processing. The overall growth rate for the two clutches used in this study was ∼0.25 mm SL/day (Fig. 11). This rate is comparable to the overall growth rate in an analysis of cranial ossification in zebrafish raised at 28.5°C (y = 0.25x + 2.56; Cubbage and Mabee, 1996).

Figure 11.

Size vs. age for TAB-5 fish reared for this study (y = 0.253x + 2.829, n = 202). Flexion occurred at 5.5 mm standard length.

Additional adult fish (EK strain, wild-type) were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Several adult Tubingen strain wild-type fish were fixed in 10% formalin in PBS. Finally, two offspring of wild-caught fish (courtesy of Dr. Amy McCune, Cornell University) were fixed in 10% formalin in PBS at room temperature for comparison with wild-type lab strains.

All fishes used in this study were killed with MS222 or cooled on ice using standard methods. Because development of the lateral line system with fish size and not age (Münz, 1986; Higgs et al., 2001), standard length (SL) is recorded for all material used in this study. Nevertheless, actual or approximate ages (in dpf) based on a regression of SL vs. days postfertilization (Fig. 11) are provided for all study material.


An ontogenetic series of TAB-5 fish (n = 13, 2–80 dpf; 8.5–22 mm SL, in 0.5- or 1.0-mm increments) and one EK (30 mm SL) were prepared histologically. Individuals < 6.0 mm SL were not decalcified. Individuals 6.0–7.5 mm SL were decalcified for 2 hr, individuals 8.0–8.5 mm SL were decalcified for 3.5 hr, individuals > 8.5 mm SL were bisected at the level of the pectoral fin, and heads were decalcified for 8 hr (Cal-Ex, Fisher). All specimens were washed for 3 hr in PBS at room temperature, transferred through an ascending sucrose series in PBS, dehydrated in a graded ethanol/t-butanol series, and embedded in Paraplast Plus (Fisher). Blocks were sectioned transversely at 8 μm, mounted on albumin-subbed slides and stained by using a modified HBQ stain (Hall, 1986) to differentiate bone from cartilage and soft tissue.

Scanning Electron Microscopy

Thirty-two TAB-5 wild-type fish (2–72 dpf, 3.0 mm–23 mm SL), one EK wild-type (31 mm SL) and two F1 (20 mm SL) of field-caught fish were prepared for SEM. Several of the smaller TAB-5 fish (5–9 mm SL) were sonicated to remove surface mucus to facilitate observation of neuromasts. All specimens were dehydrated in an ascending ethanol series on a shaker table, critical point dried in CO2, and mounted on carbon-coated aluminum stubs to facilitate either a view of the mandibular canal or the supraorbital canal, on the ventral and dorsal aspects of the head, respectively. The jaws of the two largest specimens were removed and processed separately to allow viewing of the mandibular and supraorbital canals in the same fish. Mounted fish were coated with gold–palladium alloy and viewed by using a Hitachi model S5-7 scanning electron microscope. Hair cell numbers were counted by using enlarged, digitized SEM images. These data should be considered to be minimum hair cell numbers, because neighboring hair cell bundles may have obscured some hair cells. Neuromast length and width and maximum kinocilium length were made with electronic caliper measurements on 4 × 5 SEM Polaroid photographs. These should be considered as minimum measurements due to preparation artifact, such as breakage of kinocilium and the internal curvature of the canals.

Clearing and Staining

An ontogenetic series of TAB-5 fish (9–32 mm SL), as well as one adult EK (32 mm SL) and several adult TUB fish were enzymatically cleared and stained for bone (alizarin red) and cartilage (Alcian blue; Pothoff, 1983) to observe patterns of cranial ossification and development of the bones containing the lateral line canals.


We thank Dr. Nancy Hopkins and Sarah Farrington (Center for Cancer Research, MIT) for generously providing the TAB-5 fish that formed the basis of this study. Dr. Amy McCune (Cornell University), Ms. Beth Linnon (MBL, Woods Hole), and Dr. Mary Mullins (University of Pennsylvania School of Medicine) provided additional material for this study. Dr. Norman Dollahon provided SEM expertise, and Jonathon Yoder assisted with analysis of neuromast morphology. J.E.S. was supported by an HHMI Undergraduate Education Grant to Villanova University.